PROJECT SUMMARY The gustatory cortex (GC) has been the subject of great attention over the past years. While its role in processing taste is well established, several issues regarding the strategy used by GC for coding gustatory information remain unsolved. The recent adoption of 2-photon calcium imaging to map the surface of GC in anesthetized mice has highlighted the possibility that gustatory information may be encoded by spatial patterns of single neuron activity. An influential proposal suggested a ?gustotopic? organization of taste coding, featuring spatially localized ?hot spots? formed by single clusters of neurons responding exclusively to a single taste quality. More recent imaging data complemented, and partially challenged this hypothesis, by showing spatially distributed representations in portions of GC between hotspots. Alas, both the studies published so far were performed in anesthetized animals. The impervious anatomical location of GC has hindered the application of 2- photon calcium imaging to study the topographical organization GC in alert mice. As a result, the nature of spatial coding in alert animals remains unknown. The research proposed in this grant will establish the experimental protocols for imaging large ensembles of neurons from the surface of GC in alert mice. The proposed research focuses on the following aims: Aim #1 will establish the methods for 2-photon calcium imaging using microprisms implanted on the surface of GC in mice licking for different tastants; Aim #2 will perfect the ability to monitor activity in the same ensemble for multiple days; Aim #3 will combine calcium imaging with behavioral training to monitor neural activity in mice engaged in taste discriminations. In addition to achieving technical milestones, the experiments in each aim will also address important scientific questions. Specifically, Aim #1 will investigate spatial patterns of activity on the surface of GC, addressing how wakefulness affects gustotopy and introduces temporal dynamics. Aim #2 will investigate the balance between plasticity and stability in GC representations, addressing how familiarization and repeated exposure affect taste evoked activity. Finally, Aim #3 will unveil the relationship between spatio-temporal patterns of activity and perception, by imaging GC in mice learning and performing simple and difficult discriminations in a taste-based, two alternative forced choice task. Altogether, the proposed experiments will answer fundamental questions in the field of gustation and open an entirely new research pipeline for future studies. Future studies made possible by this approach will investigate GC responses to multiple stimuli (multiple tastants, odors, flavors and cross-modal cues), unveil whether anticipatory cues can recruit taste representations and investigate coding in different cell types.